Intestinal Pgc1α ablation protects from liver steatosis and fibrosis

Background & Aims The gut–liver axis modulates the progression of metabolic dysfunction-associated steatotic liver disease (MASLD), a spectrum of conditions characterised by hepatic steatosis and a progressive increase of inflammation and fibrosis, culminating in metabolic dysfunction-associated steatohepatitis. Peroxisome proliferator-activated receptor-gamma coactivator 1α (Pgc1α) is a transcriptional co-regulator of mitochondrial activity and lipid metabolism. Here, the intestinal-specific role of Pgc1α was analysed in liver steatosis and fibrosis. Methods We used a mouse model in which Pgc1α was selectively deleted from the intestinal epithelium. We fed these mice and their wild-type littermates a Western diet to recapitulate the major features of liver steatosis (after 2 months of diet) and metabolic dysfunction-associated steatohepatitis (after 4 months of diet). The chow diet was administered as a control diet. Results In humans and mice, low expression of intestinal Pgc1α is inversely associated with liver steatosis, inflammation, and fibrosis. Intestinal disruption of Pgc1α impairs the transcription of a wide number of genes, including the cholesterol transporter Niemann–Pick C1-like 1 (Npc1l1), thus limiting the uptake of cholesterol from the gut. This results in a lower cholesterol accretion in the liver and a decreased production of new fatty acids, which protect the liver from lipotoxic lipid species accumulation, inflammation, and related fibrotic processes. Conclusions In humans and mice, intestinal Pgc1α induction during Western diet may be another culprit driving hepatic steatosis and fibrosis. Here, we show that enterocyte-specific Pgc1α ablation protects the liver from steatosis and fibrosis by reducing intestinal cholesterol absorption, with subsequent decrease of cholesterol and de novo fatty acid accumulation in the liver. Impact and implications Liver diseases result from several insults, including signals from the gut. Although the incidence of liver diseases is continuously increasing worldwide, effective drug therapy is still lacking. Here, we showed that the modulation of an intestinal coactivator regulates the liver response to a Western diet, by limiting the uptake of dietary cholesterol. This results in a lower accumulation of hepatic lipids together with decreased inflammation and fibrosis, thus limiting the progression of liver steatosis and fibrosis towards severe end-stage diseases.


Impact and implications
Liver diseases result from several insults, including signals from the gut.Although the incidence of liver diseases is continuously increasing worldwide, effective drug therapy is still lacking.Here, we showed that the modulation of an intestinal coactivator regulates the liver response to a Western diet, by limiting the uptake of dietary cholesterol.This results in a lower accumulation of hepatic lipids together with decreased inflammation and fibrosis, thus limiting the progression of liver steatosis and fibrosis towards severe end-stage diseases.

Introduction
Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly termed non-alcoholic fatty liver disease (NAFLD), represents the world's leading cause of chronic liver disease, with more than 25% of the global population affected. 1his number is proportionally increasing with the raising of metabolic syndrome, obesity, insulin resistance, and diabetes mellitus type 2. Hepatic fat accumulation is the sign of MASLD, which may progressively lead to liver dysfunctions, inflammatory cell infiltration, and scarring peculiar to metabolic dysfunction-associated steatohepatitis (MASH), formerly termed non-alcoholic steatohepatitis (NASH), with a crescendo towards advanced liver diseases, cirrhosis, and hepatocellular carcinoma.However, despite the efforts made to characterise these diseases, MASLD and its sequelae remain without effective drug treatment.Bariatric surgery often remains the last therapeutic option for individuals with morbid obesity and MASH. 2 Hepatic steatosis is a well-recognised hallmark of MASLD, and it is caused by the build-up of different lipid species in the liver, including triglycerides and cholesterol.An excess of triglycerides may derive from increased adipose tissue lipolysis, de novo lipogenesis, or a nutritional overload.However, although triglycerides are the most abundant hepatic lipid species, they represent a 'safe' storage solution in the liver. 3By contrast, an overabundance of cholesterol has deleterious hepatic effects, culminating with the so-called cholesterol-associated steatohepatitis (CASH). 3When the synthesis or uptake of cholesterol is increased and/or the cholesterol excretion is reduced, cholesterol starts to accumulate within the hepatocyte's lipid droplets.By inhibiting the circulating proprotein convertase subtilisin/kexin type 9 (PCSK9), an increased expression of the LDL receptor (LDLR) that facilitates the LDL uptake occurs. 4This exposes the liver to a considerable amount of cholesterol that, on one side starts to crystalise within hepatocyte lipid droplets, driving necroinflammation and liver dysfunctions, 4 and on the other, blocks the proteasomal degradation of TAZ, promoting fibrosis. 5verall, this results in a higher risk of steatohepatitis and liver cancer.
Recent investigations have postulated a crucial role of the gut-liver axis in the promotion of metabolic liver diseases. 6][8] Peroxisome proliferator-activated receptor gamma coactivator 1a (Pgc1a) was first described as a coactivator involved in the promotion of metabolic pathways especially under conditions of energy deprivation.Mostly expressed in highly metabolic organs, Pgc1a regulates the expression of genes involved in mitochondrial metabolism, antioxidant response, gluconeogenesis, and fatty acid b-oxidation. 9In the gut, Pgc1a regulates the apoptotic processes that physiologically take place at the tip of the villi. 10,11In Drosophila melanogaster, the intestinal overexpression of the Pgc1a homologue is essential to modulate gut permeability, maintaining homoeostasis and prolonging the lifespan. 12In line with this, disruption of Pgc1a expression has been associated with colitis and colorectal cancer in both humans and mice. 11,13However, whether the ablation of Pgc1a in the intestine can promote liver steatosis and fibrosis onset has not been investigated so far.
Here, we take advantage of an engineered mouse model to explore whether intestinal Pgc1a is crucial in the development of hepatic steatosis and fibrosis.Surprisingly, we found that the lack of Pgc1a from the gut impairs cholesterol absorption, finally antagonising the onset of liver steatosis and the progression to fibrosis.

Animals
Mice were kept in a pathogen-free facility, at 21 ± 2 C with a 12h light/dark cycle, and had free access to food and water.All the murine strains we used were in C57BL6/J background.To generate iPgc1a -/-mice, Pgc1a fl/fl mice 14 were intercrossed with Vil1-Cre mice (Jackson Laboratory, Bar Harbor, ME, USA) to obtain Vil1-Cre Tg/-Pgc1a fl/-mice.These mice were then backcrossed with Pgc1a fl/fl mice to restore homozygosity for the iPgc1a floxed allele.Male Vil1-Cre Tg/-Pgc1a fl/fl mice were bred to Pgc1a fl/fl mice to produce the mice used in the study.Pgc1a fl/fl mice were used as controls.Eight-week-old mice were treated for 8 or 16 weeks with 42% kcal/fat diet enriched with saturated fats (0.2% total cholesterol, milk fat) (Teklad, TD.180342).Chow diet was used as a control.Mice were randomly assigned to treatment groups for in vivo studies.Food intake and body weight were monitored weekly.All mice were sacrificed randomly after overnight fasting at Zeitgeber Time (ZT) 3.Each animal experiment was repeated using at least two cohorts of mice.All the experiments were performed according to the ethical protocol authorised by the Italian Ministry of Health (n.1208/2020-PR).

Results
Intestinal Pgc1a is induced in liver steatosis and fibrosis To investigate whether there is any correlation between MASLD and intestinal Pgc1a modulation, we analysed the mRNA expression of Pgc1a and its target genes in mice after the induction of liver steatosis and MASH.Pgc1a levels were significantly increased in the intestines of mice fed a Western diet (WD) for either 8 weeks (Fig. 1A and B) or 16 weeks (Fig. 1C and  D).Whereas in the chow diet-fed mice the expression of the coactivator is mainly localised at the tip of the villi, as previously described, 11 in WD-fed mice Pgc1a is more scattered along the crypt-villus axis (Fig. 1B and D).In humans, obesity is frequently associated with liver steatosis and related complications.As bariatric surgery (Roux-en-Y gastric bypass) in grossly obese individuals is a way to decrease hepatic steatosis, inflammation, and fibrosis, 2 we analysed the transcriptional profile of jejunal biopsies from obese patients before and 1 month after gastric bypass (GSE113819), observing a significant downregulation of PPARGC1A expression 15 (Fig. S1A and B).Therefore, we may infer a negative correlation between the level of Pgc1a expression and the extent of liver steatosis and fibrosis in both mice and humans.

Intestinal-specific Pgc1a ablation affects gene expression
To explore the role of intestinal Pgc1a in the development of liver disorders, we generated iPgc1a -/-mice by crossing Pgc1a fl/fl mice with mice expressing Cre recombinase under Villin promoter to drive a specific intestinal deletion of exons 3-4-5 of Pgc1a gene (Fig. 1E).The specific deletion was confirmed by reversetranscription quantitative PCR (RT-qPCR) for Pgc1a in the different intestinal tracts (Fig. 1F).RT-qPCR revealed a lower ileal expression of Pgc1a target genes Mitochondrial transcription factor A (Tfam), Cytochrome C (Cyt-C), and ATP synthase F1 subunit beta (Atp5b) in iPgc1a -/-mice compared with Pgc1a fl/fl littermates (Fig. 1G).Moreover, the ablation of Pgc1a determined a reduction in the mitochondrial endogenous, uncoupled, and Cox respiratory capacities in freshly isolated intact enterocytes (Fig. 1H).No difference in the expression of Pgc1a and its target genes was detected in the liver, white adipose tissue (WAT), and quadriceps by RT-qPCR (Fig. 1I-K).
To mimic the onset of MASLD, 2-month-old iPgc1a -/-and Pgc1a fl/fl littermates were fed a WD for 2 months.A regular chow diet was used as a control.No major modifications were detected in the intestinal architecture of the two genotypes at the time of sacrifice (Fig. 2A).Although a decreased length of intestinal villi was detected in iPgc1a -/-mice fed a chow diet, this difference became inconsistent following WD feeding (Fig. 2B).
To identify molecular pathways regulated by Pgc1a in response to WD, microarray analysis was performed on the ileum cells harvested from iPgc1a -/-and Pgc1a fl/fl mice after 2 months of diet.The principal component analysis of the transcriptome revealed that diet is the principal component affecting gene expression, followed by the genotype (Fig. 2C).Among the differentially expressed genes in the comparison between the two diet treatments, up to 53% (993 genes) were common to both genotypes, whereas 25% (460 genes) and 22% (402 genes) were differentially expressed in Pgc1a fl/fl and iPgc1a -/-mice, respectively (Fig. 2D).Hierarchical clustering analysis of genes affected by WD in both genotypes identifies five clusters (Fig. 2E).Genes from clusters 2 and 5 were sensitive to the diet but not dependent on Pgc1a expression (Fig. 2F).Genes from cluster 4 display a difference between the two genotypes under only the chow diet regimen.The lack of Pgc1a altered the expression of genes in both diet conditions (clusters 1 and 3).
Genes in cluster 1 are mainly induced in iPgc1a -/-animals and enriched for autophagy pathways.Intriguingly, genes in cluster 3 showed a decreased expression in iPgc1a -/-mice compared with Pgc1a fl/fl mice, with a marked downregulation as a result of WD consumption.Gene enrichment analysis revealed that genes of this cluster are mostly involved in the regulation of gene expression and translation (Fig. 2F).
Occludin (Ocln) in iPgc1a -/-mice compared with controls in the chow diet, inconsistencies were detected between the two genotypes after WD administration (Fig. S2B).To further investigate whether the deletion of Pgc1a from the gut unbalances intestinal integrity, we measured plasma levels of FITCconjugated dextran (Fig. S2C) and of lipopolysaccharide-binding protein (Fig. S2D), finding no differences in intestinal permeability between iPgc1a -/-and Pgc1a fl/fl mice in both diet conditions.
Intestinal-specific Pgc1a ablation protects against liver steatosis After 2 months of WD, iPgc1a -/-mice displayed less body weight gain than Pgc1a fl/fl mice with comparable food consumption and a significantly lower liver-to-body weight ratio after WD feeding (Fig. 3A and B, and Fig. S3).The WAT-to-body weight ratio was affected by WD consumption, but not by genotype (Fig. 3C).Comparable levels of circulating triglycerides and cholesterol were detected among the two genotypes (Fig. 3D and E).H&E and Oil Red O staining showed a decreased lipid accumulation in iPgc1a -/-mice compared with controls (Fig. 3F), consistent with a lower steatosis score and a decreased accumulation of lipid specimens (triglycerides, and total and esterified cholesterol) within the liver (Fig. 3G and H).Given that reduced insulin sensitivity is frequently associated with MASLD, we assessed the glucose response in our mice.No remarkable changes in insulin sensitivity were detected between the two genotypes in both diet conditions or in the level of plasmatic incretin glucagon-like peptide 1 (Glp-1) (Fig. S4A-E).
To evaluate whether the lower steatosis observed in iPgc1a -/- mice was caused by changes in the synthesis of new fatty acids, we assessed the expression of Sterol regulatory element-binding protein 1 (Srebp1c), Fatty acid synthase (Fasn), and Stearoyl-CoA desaturase 1 (Scd1), observing a significantly decreased expression of the de novo lipogenesis genes in iPgc1a -/-mice compared with controls (Fig. 3I).No difference was detected in the expression genes responsible for de novo fatty acid synthesis in WAT (Fig. S5A), thus indicating the involvement of a specific hepatic mechanism.Once absorbed by the intestine, fatty acids are primarily stored in the WAT, from where they are released during lipolysis induced by fasting, driving a specific hepatic transcriptional response. 16However, the expression levels of Adipose Triglyceride Lipase (Atgl), Hormone-Sensitive Lipase (Hsl), and Lipoprotein Lipase (Lpl) were not different between the two genotypes and are mainly modulated by diet consumption (Fig. S5B).Furthermore, to assess whether increased consumption of fatty acids by the muscle could be responsible for the observed phenotype, we measured the mRNA levels of fatty acid b-oxidation-related genes in the quadricep of our mice, finding no changes (Fig. S5C).
Next, we explored whether modifications of genes related to cholesterol metabolism were responsible for the decreased cholesterol accumulation observed.The expression of Ldlr and Scavenger receptor class B type (Scarb1), two major cholesterol importers, was mainly affected by genotype (Fig. 3J): whereas Ldlr levels were slightly induced in iPgc1a -/-mice in both diet conditions, the induction of Scarb1 by the WD regimen was abolished in iPgc1a -/-mice as opposed to Pgc1a fl/fl mice.The mRNA levels of 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr), codifying for the rate-limiting enzyme of cholesterol synthesis, were affected only by the diet, but not by the genotype (Fig. 3K).Finally, the expression of genes involved in cholesterol excretion from the hepatocytes, ATP-binding cassette G5 and G8 (Abcg5/8), was significantly reduced in iPgc1a -/-mice after WD (Fig. 3L).Altogether, this suggests that the intestinal-specific Pgc1a ablation protects against liver steatosis via modulation of liver X receptor (Lxr)-driven cholesterol and fatty acid metabolism.However, the mRNA level of Lxra and Lxrb did not reveal any alteration between the different groups (Fig. 3M), suggesting that the activity of Lxrs, rather than the expression, is involved in the observed phenotype.Indeed, as Srepb1c, Fasn, and Abcg5/8 are all targets of Lxrs, 17,18 one could speculate that intestinal Pgc1a ablation decreased hepatic Lxr transcriptional activation via a reduction of their ligands, the cholesterol derivatives oxysterols.

Intestinal-specific Pgc1a ablation protects against steatohepatitis
To investigate whether the intestinal-specific Pgc1a ablation was still able to keep a protective phenotype in hepatic MASH, we sacrificed the mice after 4 months of diet.Consistently with steatosis data, iPgc1a -/-mice fed a WD displayed lower body weight and liver-to-body weight ratio, whereas the WAT-to-body weight ratio was influenced by only the diet (Fig. 4A-C).Moreover, the plasma alanine aminotransaminase level, a recognised marker of liver damage, was less elevated in iPgc1a -/-mice than in Pgc1a fl/fl mice in the WD condition (Fig. 4D).
Inflammation and fibrosis represent two hallmarks of MASH. 6e first examined the distribution of liver macrophages with F4/ 80 immunostaining, a representative macrophage marker (Fig. 4E, upper panel).No considerable staining was detected in the liver of mice fed with a chow diet.In iPgc1a -/-mice, macrophages showed a scattered distribution in the liver section.In contrast, in Pgc1a fl/fl mice fed a WD, macrophages aggregated to surround hepatocytes with large lipid droplets, forming the hepatic crown-like structure (hCLS).Importantly, the hCLS represents a histological feature reflecting the extent of activation of Kupffer cells and liver fibrosis, 19 and their number was lower in the liver of iPgc1a -/-mice than in the liver of controls fed a WD (Fig. 4F), thus reflecting a diminished inflammatory and fibrotic process.In line with this, the expression of the macrophage surface marker Cd68 was slightly decreased in iPgc1a -/-mice compared with Pgc1a fl/fl mice after WD feeding (Fig. 4H).Moreover, we observed a reduced mRNA level of M1 macrophage markers C-C motif chemokine ligand 2 (Ccl2) and tumor necrosis factor a (Tnfa) in iPgc1a -/-mice compared with control littermates (Fig. 4I).No differences were detected for interleukin1b (Il1b), whose expression is negatively affected by Lxr activity, 20 supporting once more our observations regarding the reduction of Lxr activation in iPgc1a -/-mice.The analysis of M2 macrophage markers (Cd206 and Arginase 1, Arg1) revealed a trend to increase in iPgc1a -/-mice compared with controls, although not significant (Fig. 4J).A trend towards reduction in the expression of the hepatic stellate cell inducer transforming growth factor-beta (Tgfb) was detected in iPgc1a -/-mice compared with control littermates.To understand whether a different macrophage polarisation may be involved in our phenotype, we calculated the ratio between M1 and M2 macrophage markers (Fig. 4K).Interestingly, we observed that whereas Pgc1a fl/fl mice displayed an increased macrophage polarisation towards the M1 phenotype, usually associated with inflammation, iPgc1a -/-mice showed a significant reduction in the M1/M2 ratio, suggesting the occurrence of a more anti-inflammatory phenotype.Then, to dissect fibrosis, we examined collagen deposition in the liver of our mice.iPgc1a -/- mice fed a WD presented lower fibrosis than the control counterpart, as indicated by the Sirius Red staining (Fig. 4E and G) and the reduced mRNA level of genes involved in fibrosis (Fig. 4L).Overall, these data demonstrated that the intestinal-specific Pgc1a ablation confers protection also against liver fibrosis and inflammation.(G) Fibrosis quantified as the percent surface area occupied by SR-stained collagen.Hepatic relative mRNA expression of (H) Cd68 (I) M1 macrophage markers, and (J) M2 macrophage markers.(K) M1/M2 ratio.(L) Hepatic relative mRNA expression of genes involved in fibrosis.All the experiments were performed on 6-month-old iPgc1a -/-and Pgc1a fl/fl littermates fed a CD or WD for 4 months (n = 8 animals/group).Data are expressed as mean ± SEM.Comparison between different groups was performed using two-way ANOVA followed by Sidak's multiple comparison tests; *genotype effect, # diet effect (* or # p <0.05; ** or ## p <0.01; *** or ### p <0.001).Acta2, actin alpha 2; ALT, alanine aminotransferase; Arg1, Arginase 1; BW, body weight; Ccl2, C-C motif chemokine ligand 2; Col1a1, collagen type I alpha 1 chain; hCLS, hepatic crown-like structure; I1b, interleukin 1b; LW, liver weight; MASH, metabolic dysfunction-associated steatohepatitis; Mmp 9/13, matrix metalloproteinase 9/13; Pgc1a, peroxisome proliferator-activated receptor-gamma coactivator 1a; SR, Sirius Red; Tgfb, transforming growth factor-beta; Tnfa, tumor necrosis factor a; WD, Western diet; WW, white adipose tissue weight.

Intestinal-specific Pgc1a ablation does not impair intestinal fatty acid absorption
The results obtained prompted us to investigate whether an impaired absorption of fatty acids driven by the absence of Pgc1a may explain the protective hepatic phenotype.To this end, we assessed the expression of the major fatty acid transporters in the intestine (Fig. 5A).WD feeding increases the mRNA levels of all the genes evaluated.Although no differences were observed for Fatty Acid Translocase (Fat/Cd36), Fatty Acid Binding Protein 1 (Fabp1), and Microsomal Triglyceride Transfer Protein (Mttp) between the two genotypes, the mRNA levels of Fatty Acid Transporter 4 (Fatp4) were significantly decreased in iPgc1a -/- mice fed a WD compared with controls.However, the oral lipid tolerance test did not display any difference in fatty acid absorption among the two genotypes in both diets (Fig. 5B).
Moreover, the intestinal triglyceride level difference is comparable between the groups (Fig. 5C).A possible explanation may reside in the low expression of fatty acid b-oxidation genes driven by the absence of Pgc1a (Fig. 5D).Finally, we checked the level of genes involved in the de novo lipogenesis process (Srebp1c, Fasn, and Scd1), but the main differences observed were attributable more to the diet than to the genotype (Fig. 5E).Therefore, we may assume that Pgc1a does not affect intestinal fatty acid absorption.
in intestinal cholesterol trafficking.To this end, we measured the expression of genes regulating both cholesterol excretion and uptake in the intestine.Specifically, we analysed Abcg5/8, codifying for the sterol transporters that prevent the accumulation of dietary sterols, and ATP-binding cassette A1 (Abca1), which directly mediates cholesterol transport towards plasma HDL.The expression of all those genes was affected by WD regimen, but not by genotype (Fig. 6A).On the contrary, the level of Scarb1 was diminished only in the enterocytes of iPgc1a -/-mice fed a chow diet compared with control mice, whereas no changes were detected during WD (Fig. 6B).As Scarb1 does not cause overall intestinal cholesterol absorption in vivo, 21 we analysed Niemann-Pick C1-like 1 (Npc1l1) mRNA level, which translates for a cholesterol transporter in the small intestine, finding a statistically significative reduction of this mRNA in iPgc1a -/-mice fed a WD compared with controls (Fig. 6C).No difference was detected in the intestinal cholesterol content, suggesting similar cholesterol retention between the two genotypes (Fig. 6D).To further validate this observation, we directly assessed the absorption of exogenous cholesterol by measuring serum fluorescence following oral delivery of a fluorescently labelled cholesterol mimetic.The ablation of Pgc1a correlates with a significantly decreased serum fluorescence after 8 h from administration (Fig. 6E), thus indicating an impaired cholesterol absorption in iPgc1a -/-mice.

Discussion
A critical aspect in the pathogenesis of hepatic steatosis is the development of inflammatory and fibrotic processes, which facilitate the progression towards severe liver diseases, such as cirrhosis and cancer.
The accumulation of harmful lipids is one of the factors that predispose the liver to develop steatohepatitis and its sequelae.It is worth noting that not all the classes of lipids drive lipotoxicity mechanisms, intimately associated with the establishment of chronic inflammation. 6Excessive availability of cholesterol in the hepatocytes has deleterious effects, promoting cell damage and activating the fibrotic pathway. 4,5lthough cholesterol can be endogenously synthesised, this process is disadvantageous for the cell, as it requires a considerable energetic expense.By contrast, large amounts of cholesterol can be easily accessed from the diet.In this view, the small intestine plays a unique role in cholesterol homoeostasis, 22 by regulating cholesterol absorption and excretion.Npc1l1 is the rate-limiting transporter of cholesterol in the small intestine, thus representing the pivotal regulator of cholesterol uptake from the gut lumen andmore in generalof systemic cholesterol homoeostasis.In humans, NPC1L1 is mainly expressed in the liver and the gut, but in murine models, its expression is limited to the gut. 23Therefore, modifications in the activity of this transporter are directly attributable to the intestine.
Ezetimibe is an antihypercholesterolaemic drug that disrupts the structural cluster formed by NCP1L1 and cholesterol, thus leading to the inhibition of NPC1L1 functions. 24Both genetic and pharmacological inactivation of Npc1l1 counteract the development of hepatic steatosis in mice fed high-fat diets, by decreasing the amount of hepatic cholesterol. 25,26Moreover, also in humans, the ezetimibe treatment improves hepatic steatosis and inflammation in either obese individuals or non-obese ones with MASH, [27][28][29] although the results are not consistent with other clinical trials. 30n the current study, we found that an increased intestinal Pgc1a expression is associated with hepatic lipid accumulation, All the experiments were performed on 4-month-old iPgc1a -/-and Pgc1a fl/fl littermates fed a chow diet or WD for 2 months (n = 6-10 animals/group).Data are expressed as mean ± SEM.Comparison between different groups was performed using two-way ANOVA followed by Sidak's multiple comparison test or using Mann-Whitney U test; *genotype effect, # diet effect (* or # p <0.05; ** or ## p <0.01; *** or ### p <0.001).Abca1, ATP-binding cassette A1; Abcg5/8, ATP-binding cassette G5/G8; Npc1l1, Niemann-Pick C1-like 1; Pgc1a, peroxisome proliferator-activated receptor-gamma coactivator 1a; Scarb1, scavenger receptor class B type; WD, Western diet.inflammation, and fibrotic processes in both humans and mice, thus priming the progression of metabolic liver diseases.The generation of a mouse model in which Pgc1a is specifically deleted from the gut reveals that the ablation of the coactivator does not impair intestinal architecture or gut permeability.Although this may appear contradictory with a previous study in which the expression of Pgc1a homologue was necessary to regulate intestinal integrity in old flies, 12 it has to be considered that our work was carried out in mice not older than 4-6 months and that ageing may be a crucial aspect to keep in consideration.
When we challenged the mice with a WD, we observed a dramatic reduction of hepatic steatosis, as a result of the lower accumulation of cholesterol and triglycerides in the hepatocytes (Fig. 3).Consistently, prolonged exposure to the diet results in lower hepatic inflammation and fibrosis, two aspects of MASH.Importantly, these effects seem to be driven specifically by the gut, given that we did not detect any alteration in systemic lipid disposal, as de novo lipogenesis or lipolysis processes in the WAT or altered fatty acid oxidation in the muscle.Remarkably, whereas we did not find any difference in the intestinal uptake of fatty acids, we observed a significant reduction of Npc1l1 in the small intestine of animals lacking Pgc1a fed a WD, which led to an impaired cholesterol absorption (Fig. 6).The expression of Npc1l1 is regulated by different transcription factors and nuclear receptors, including the Peroxisome proliferator-activated receptor a (Ppara), the Sterol regulatory element-binding protein 2 (Srebp2), and the Hepatocyte Nuclear Factor 4a (Hnf4a).Using HepG2 immortalised cell line, it has been demonstrated that Npc1l1 expression can be induced either by Ppara/Retinoid X receptor-a (Rxra) dimer or by Srebp2/Hnf4a dimer. 31,32It is well recognised that Pgc1a acts as a coactivator of both Ppara and Hnf4a. 33,34And, indeed, Pgc1a boosts the activity of Ppara/Rxra and Srebp2/Hnf4a, stimulating the activation of Npc1l1 transcription. 32Although we did not assay these specific interactions in our study, this mechanism appears the most reliable one.Indeed, in our intestinal cells, we detected a significantly decreased expression of Ppara-regulated genes involved in fatty acid oxidation (Fig. 5D).However, we did not observe a reduction of Fabp1 gene expression, as recently described, thus suggesting that probably other cofactors are needed to regulate that pathway. 7everal pieces of evidence demonstrated that dietary cholesterol promotes the hepatic build-up not only of cholesterol but also of triglyceride. 35Our findings on iPgc1a -/-mice are in line with these observations.Indeed, the decreased cholesterol uptake from the gut results in a significant reduction of triglyceride accumulation in the liver as a result of a lower synthesis of new fatty acids.To note, ezetimibe treatment protects from MASLD by inhibiting Srebp1c, the master regulator of de novo lipogenesis. 368][39] In our model, the expression of Lxrs target genes related to de novo lipogenesis as well as cholesterol secretion in the liver is impaired, thus corroborating the idea that the lack of Pgc1a in the gut disrupts the intestinal Npc1l1 expression and reduces intestinal cholesterol absorption and subsequent hepatic lipid content via downregulation of Lxr transcriptome.
Recently, various compounds that target Pgc1a have been developed, and the beneficial effects on different conditions have been effectively evaluated.Among them, the small inhibitor SR-18292 improved the metabolic outcome of diabetes type 2, a condition frequently associated with MASLD and its sequelae, by blocking the gluconeogenic pathway and, more in particular, displacing the interaction between Pgc1a and Hnf4a. 40Although the effects of this drug on the intestine have not been tested yet, it would be interesting to see whether they can disrupt the interaction between Pgc1a and Hnf4a also in the enterocytes, thus leading to lower Npc1l1 expression.
In humans and mice, intestinal Pgc1a induction may be another culprit that drives WD-mediated liver steatosis and fibrosis.In the present study, we showed that enterocytes' specific ablation of Pgc1a protects from hepatic steatosis and fibrosis driven by the WD via a reduction of intestinal cholesterol absorption, and subsequent decrease of cholesterol and de novo fatty acid accumulation in the liver.

Abbreviations
AND "small intestine" AND "homo sapiens", recovering 7 different datasets.Datasets with specimens from other organisms, expression profiles by RT-qPCR or RNA sequencing, or the absence of a clear control group were excluded.

Microarray Analysis
Microarray data were analyzed using R and Bioconductor packages [5].Raw data (median signal intensity) were filtered, log2 transformed, corrected for batch effects (microarray washing bath and labelling serials), and normalized using the qsmooth method [6].A model was fitted using the limma lmFit function [7].Pairwise comparisons between biological conditions were applied using specific contrasts.A correction for multiple testing was applied using the Benjamini-Hochberg procedure to control the false discovery rate (FDR).Probes with an FDR≤0.05 were considered to be differentially expressed between conditions.Hierarchical clustering was applied to the samples and the differentially expressed probes using 1-Pearson correlation coefficient as distance and Ward's criterion for agglomeration.
The clustering results are illustrated as a heatmap of expression signals.Gene ontology and transcription factor enrichment analysis were performed using Metascape [8].

Histology and Immunohistochemistry
Tissue specimens were fixed in 10% formalin for 12-24 hours, dehydrated, and paraffinembedded.Ileum and liver sections (2 μm) were stained with hematoxylin-eosin staining (HE), according to the standard procedures.Villi length was calculated by evaluating complete, full-sized intestinal villi (n=10) not exhibiting bending or mechanical damage, for each sample.Sirius Red staining using Direct Red 80 and Fast Green FCF (Sigma-Aldrich, USA) was performed on liver sections to assess fibrosis.Immunohistochemistry analysis was performed in liver and ileum specimens (4 μm).Briefly, sections were subjected to antigen retrieval by boiling the slides in sodium citrate pH 6 for 15 minutes, permeabilized in phosphate-buffered saline with 0.25% Triton X-100 for 5 minutes, and then incubated for 10 minutes at room temperature in protein blocking solution (Dako, Denmark).Subsequently, sections were incubated with primary antibodies as indicated in Supplementary Table 2.
Sections were washed in PBS for 15 minutes and incubated at room temperature with DAKO real EnVision detection system (Dako, Denmark), according to the manufacturer's instruction.For negative controls, 1% nonimmune serum in PBS substituted the primary antibodies.Images were acquired and analyzed with Aperio Image Scope (Leica Biosystems, Germany).The percentage of stained area/total area was evaluated in 10 consecutive acquired images.Values from all consecutive images for each sample were averaged and displayed as mean±SEM.Steatosis score was assigned based on the percentage of hepatic parenchyma containing fat: 0 -<5%; 1 -5-33%; 2 -33-66%; 3 ->66% [9].Specimens from livers were embedded in OCT (Sakura), frozen under nitrogen vapours, and stored at -80°C.Liver cryosections (4 μm) were stained with Oil Red O Stain Kit (ab150678, Abcam, UK) following the manufacturer's indications.

Lipidomic Assay
Fatty acids were extracted from frozen tissues or plasma using a modified Bligh and Dyer extraction method.Samples were lysed in a water EDTA (5 Mm)/methanol mix (1:2 vol/vol).
Methanol and dichloromethane were added to reach the following ratios of MeOH/water/CH2Cl2: 2.5/2.0/2.5.Glyceryltrinonadecanoate was added as an internal standard.The dried lipid extract was transmethylated with 1 ml of BF3 in methanol (1:20, vol/vol) for 60 min at 100°C, evaporated to dryness, and the fatty acid methyl esters (FAMEs) were extracted with hexane/water (3:1).The organic phase was evaporated to dryness and dissolved in 50 μl ethyl acetate.FAMEs were analyzed by gas-liquid chromatography on a 5890 Hewlett-Packard system using a Famewax fused-silica capillary column (30 m, 0.32 mm i.d., 0.25-mm film thickness; Restek).The oven temperature was programmed from 110ºC to 220°C at a rate of 2°C/min, and the carrier gas was hydrogen (0.5 bar).The injector and the detector were at 225ºC and 245°C, respectively.Neutral lipids were extracted from plasma, liver or intestine frozen tissues using a Bligh and Dyer extraction method: samples were homogenized in methanol/5 mM EGTA (2:1, v/v), and lipids (corresponding to an equivalent of 2mg tissue) extracted according to the Bligh-Dyer method63, with chloroform/methanol/water (2.5:2.5:2v/v/v), in the presence of the following internal standards: glyceryl trinonadecanoate, stigmasterol, and cholesteryl heptadecanoate (Sigma-Aldrich).Triglycerides, free cholesterol, and cholesterol esters were analyzed by gas-liquid chromatography on a Focus Termo Electron system equipped with a Zebron-1 Phenomenex fused-silica capillary column (5 m, 0.25 mm i.d., 0.25 mm film thickness).The oven temperature was programmed to increase from 200 to 350 °C at 5 °C/min, and the carrier gas was hydrogen (0.5 bar).The injector and detector temperatures were 315 °C and 345 °C, respectively.

Statistical Analysis
All the results are expressed as mean ± SEM.Statistical analyses were performed with GraphPad Prism software analysis (v9.0, GraphPad Software, USA).Outliers were calculated with ROUT or Grubbs test.To compare two groups Mann Whitney U test was used, while for four groups 2-Way ANOVA followed by Tukey's post-hoc test.A p-value <0.05 was considered significant.Paired T-Test was used to analyze paired data.Significant genotype effect was indicated by * (*p < 0.05, **p < 0.01, ***p < 0.001).Significant diet effect was indicated by # ( # p < 0.05, ## p < 0.01, ### p < 0.001).

(
B) Villi length assessed on 10 single complete full-size villi per sample.(C) PCA plots of the whole transcriptomic dataset in the intestine.Each dot represents an observation (animal) projected onto the first (horizontal axis) and second (vertical axis) PCA variables.(D) The number of genes differentially expressed between the two diets in Pgc1a fl/fl and iPgc1a -/-mice (adj.p <0.05, FC >1.5).(E) Heatmap showing data of microarray analysis on intestinal specimens.The hierarchical clustering identifies five different clusters.(F) Representation of the mean cluster profiles, the enrichment of transcription factors (TRRUST), the GO analysis, and the number of genes in each heatmap cluster.All the experiments were performed iPgc1a -/-and Pgc1a fl/fl littermates fed a chow diet or WD for 2 months (n = 6-10 animals/group).Data are expressed as mean ± SEM.Comparison between different groups was performed using two-way ANOVA followed by Sidak's multiple comparison tests; *genotype effect, # diet effect (* or # p <0.05; ** or ## p <0.01; *** or ### p <0.001).adj.p, adjusted p; FC, fold change; GO, Gene Ontology; PCA, principal component analysis; Pgc1a, peroxisome proliferator-activated receptor-gamma coactivator 1a; TRRUST, transcriptional regulatory relationships unravelled by sentence-based text-mining; WD, Western diet.